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目的:建立一种荧光可视化显像且稳定过表达缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)的裸鼠结直肠癌移植瘤模型。方法:对不同的人结直癌细胞系(SW480、SW620、LOVO及HCT116细胞)用缺氧诱导剂Co Cl2处理,根据HIF-1α被诱导表达的情况选择合适的靶细胞;将HIF-1α的c DNA序列克隆至慢病毒表达质粒p LV-TRC-EGFP,构建p LVHIF1α-EGFP慢病毒表达质粒,并包装过表达HIF-1α的慢病毒颗粒Lenti-HIF1α-EGFP。将Lenti-HIF1α-EGFP感染SW480细胞,嘌呤霉素筛选稳定过表达HIF-1α的细胞株SW480HIF-1α,Western blotting检测SW480HIF-1α细胞内HIF-1α及其下游蛋白VEGF、M1型丙酮酸激酶(pyruvate kinase expression M1,PKM1)的表达情况,Transwell实验检测其迁移能力。裸鼠腹腔注射SW480HIF-1α建立结直肠癌移植瘤模型,倒置荧光显微镜观察裸鼠腹腔移植瘤结节。结果:常氧条件下4种结直肠癌细胞均不表达HIF-1α,经Co Cl2诱导后,除SW480细胞系以外的细胞均可被诱导表达HIF-1α,故选择SW480细胞作为研究靶细胞。成功构建稳定过表达HIF-1α的细胞株SW480HIF-1α,SW480HIF-1α细胞内HIF-1α、VEGF和PKM1的表达水平均高于野生型SW480细胞,其迁移能力较野生型SW480细胞显著增强[观察视野内迁移细胞数:(250±11)vs(50±5)个,P<0.01]。SW480HIF-1α组裸鼠肠壁形成肿瘤结节数量显著多于SW480组[(15±4)vs(4±1)个,P<0.05]。结论:成功建立了高表达HIF-1α的荧光可视化裸鼠移植瘤模型,为进一步的相关功能学研究以及药物筛选提供条件。
OBJECTIVE: To establish a model of colorectal cancer xenografts in nude mice stably overexpressing hypoxia-inducible factor-1α (HIF-1α) by fluorescence visualization. Methods: Different human colorectal cancer cell lines (SW480, SW620, LOVO and HCT116 cells) were treated with hypoxia-inducible Co Cl2, and the suitable target cells were selected according to the induced expression of HIF-1α. c DNA sequence was cloned into the lentiviral expression plasmid p LV-TRC-EGFP to construct p LVHIF1α-EGFP lentiviral expression plasmid and packaged Lentiviral Lenti-HIF1α-EGFP overexpressing HIF-1α. SW480 cells were infected with Lenti-HIF1α-EGFP and SW480HIF-1α cells were stably transfected with puromycin. The expression of HIF-1α and its downstream proteins VEGF and M1 pyruvate kinase in SW480HIF-1α cells were detected by Western blotting pyruvate kinase expression M1, PKM1) were detected by Transwell assay. The nude mice were injected intraperitoneally with SW480HIF-1α to establish the model of colorectal cancer xenografts. The transplanted tumor nodules in nude mice were observed by inverted fluorescence microscope. Results: HIF-1α was not expressed in all the four colorectal cancer cells under normoxia. After CoCl2 induction, all cells except SW480 cell line could be induced to express HIF-1α, so SW480 cells were selected as target cells. The expression level of HIF-1α, VEGF and PKM1 in SW480HIF-1α and SW480HIF-1α cells successfully constructed in HIF-1α-stable cell line was significantly higher than that in wild-type SW480 cells Migration of cells within the visual field: (250 ± 11) vs (50 ± 5), P <0.01]. The number of tumor nodules in the SW480HIF-1α group was significantly more than that in the SW480 group [(15 ± 4) vs (4 ± 1), P <0.05]. CONCLUSION: Fluorescent visualized nude mice xenograft model with high expression of HIF-1α was successfully established, which provided conditions for further related functional studies and drug screening.